When blood vessels are damaged, yon Willebrand factor (vWF) is needed for platelets to recognize the damaged vascular endothelium and to form aggregates upon it. Human vWF circulates in plasma as a series of disulfide-linked multimers ranging in molecular weight from 350,000 to 1.times.6 (Girma et al., Blood 70:605 (1987)). Platelet adhesion to subendothelial components exposed by vascular damage is the first step in the platelet's response to such damage. Adhesion involves the binding of vWF to the subendothelium, and subsequent binding of platelets to vWF via a specific receptor on the platelet surface associated with platelet membrane glycoprotein Ib (GPIb). The latter is one of the major platelet membrane glycoproteins. There are approximately 25,000 copies of GPIb per platelet. GPIb contains two disulfide-linked subunits, GPIb.alpha. (Mr=145 kDa) and GPIb.beta. (Mr=24 kDa ) . Both chains appear to be integral membrane glycoproteins containing hydrophobic transmembrane domains (Clemetson et al., Proc. Natl. Acad. Sci. USA 78:2712 (1981)). The subunits are complexed with glycoprotein IX (Mr=22 kDa). Antibodies to vWF and GPIb can inhibit the adhesion response.
The exposed subendothelium is a matrix composed of collagen and elastin fibers, glycosaminoglycans, fibronectin, vWF, and other proteins. The yon Willebrand factor appears to be essential to the process by which flowing blood platelets recognize and adhere to areas of damaged endothelium. Individuals afflicted with yon Willebrand's disease, who have defective or deficient vWF, have a bleeding syndrome due to ineffective platelet-mediated hemostasis (Zimmerman and Ruggeri, Prog. Hemost. Thromb. 6, 203-236 (1982)).
Platelet adhesion to exposed endothelial components stimulates platelet secondary responses associated with shape change, thromboxane production, release of granular constituents, and exposure of other receptors on the platelet surface, such as the receptors associated with glycoprotein IIb/IIIa. These responses promote the formation of platelet aggregates and fibrin deposition at the site of injury.
Snake venom is a rich source of reagents functioning as either agonists or antagonists of the formation of platelet hemostatic plugs. Numerous platelet agonists isolated from snake venoms are known. Teng et al., Biochim. Biophys. Acta 757, 332-341 (1984) reported a protease, isolated from Vipera russellii venom, which possesses procoagulant activity. It activates Factor X in the presence of calcium and leads to platelet aggregation in platelet-rich plasma.
Thrombocytin, a thrombin-like enzyme from Bothrops atrox venom (Niewiarowski et al., Biochemistry 18, 3570-3577 (1979)), and crotalocytin, from Crotalus horridus horridus venom (Schmaier & Colman, Blood 56(6), 1020-1028, (1980)) aggregate platelets directly, probably by a mechanism similar to thrombin.
Disintegrins represent a new class of low molecular weight, RGD-containing peptides from the venoms of various snakes. They are platelet GPIIb/IIIa antagonists and potent inhibitors of platelet aggregation and fibrinogen binding to platelets. (Dennis et al., Proc. Natl. Acad. Sci. USA 87:2471 (1989); Gould et al., P.S.E.B.M. 195:168 (1990)). The disintegrin trigramin, a naturally occurring peptide purified from Trimeresurus gramineus venom, blocks the binding of fibrinogen and human vWF to the glycoprotein IIb/IIIa complex in thrombin-activated platelets (Huang et al., Biochemistry 28, 661-666 (1989)) but does not affect binding of vWF to GPIb. The motifs of disintegrin as well as metalloproteinase are present in hemorrhagic factors (isolated from T. flavoviridis venom) which are potent platelet antagonists (Takeya et al., J. Biol. Chem. 265:16068 (1990)). Albolabrin, isolated from Trimeresurus albolabris venom (Williams et al., Biochim. Biophys. Acta. 1039, 81-89 (1990)), showed a similar inhibitory activity in platelet aggregation. The biological activities of trigramin and albolabrin appear to depend upon the presence of an RGD sequence.
The effect of twenty Australian snake venoms (nineteen elapid and one hydrophid) and four crotalid venoms on human fresh and fixed platelets has been examined by Marshall & Herrmann, Thrombosis Research 54, 269-275 (1989). All venoms, except the hydrophid venom, which requires the presence of a plasma co-factor, directly caused fresh platelets to aggregate irreversibly.
Five snake venom lectins have been reported by Ogilvie et al., Thromb. Haem. 62, 704-707 (1989), which agglutinate red cells and stimulate the aggregation of human platelets. The lectin-induced platelet aggregation has been inhibited by a monoclonal antibody to GPIIb/IIIa (Ogilvie et al., Thromb. Haem. 62:704 (1989)).
Botrocetin, purified from the venom of the South American pit viper, has previously been shown to cause vWF-dependent agglutination of platelets (Andrews et al., Biochemistry 28, 8317-8326 (1989) ) . Botrocetin induces platelet agglutination by facilitating binding of vWF to GPIb, although botrocetin alone does not stimulate platelet agglutination directly (Sanders, et al., Lab. Invest. 59, 443-452 (1988)). Both vWF and a 52/48-kDa dimeric fragment of vWF bound specifically and saturably to botrocetin-coupled beads. However, glycocalicin, a proteolytic fragment of the .alpha.-chain of GPIb that contains the vWF-binding domain, did not bind to immobilized botrocetin (Id.). This agrees with the observation by Read et al., Blood 74(3) 1031-1035 (1989) that botrocetin appears to act in a two-step manner, first binding with vWF to form a complex, which then binds to GPIb to cause agglutination.
Ristocetin is an antibiotic isolated from Nocardia lurida that can cause platelet agglutination in the presence of human vWF. (Howard & Firkin, Thromb. Diath. Haemorrh., 26, 362-369 (1971)). It has been widely used as a cofactor to induce human vWF binding to platelet GPIb. Unlike botrocetin, it is not clear whether ristocetin promotes the binding of vWF to platelet GPIb by interacting with the vWF molecule or with its receptor on GPIb. Id.
Binding of human vWF to platelets in vitro occurs in the presence of ristocetin or botrocetin, whereas bovine vWF directly induces platelet agglutination. (Kirby, R. J. Lab. Clin. Med. 100:963 (1982). Bovine and human vWF bind to the same region on platelet GPIb. (Suzuki et al., Thromb. Res. 17:215 (1980)).
While the foregoing molecules are active in binding platelets and/or inducing their agglutination, they do so by a mechanism distinct from that of the hereinafter described novel polypeptides which bind GPIb close to or at the platelet binding site for vWF.